Bacteriology And Mycology Lab 2 (MIC 211) Fall 2024-2025 PDF

BACTERIOLOGY AND MYCOLOGY (MIC 211) Culture media and Bacterial staining Dr. Mahmoud Abdelaziz Dr. Mohamed Shaban Assistant professor of Microbiology Assistant lecturer of Microbiology...

BACTERIOLOGY AND MYCOLOGY (MIC 211) Culture media and Bacterial staining Dr. Mahmoud Abdelaziz Dr. Mohamed Shaban Assistant professor of Microbiology Assistant lecturer of Microbiology Date : 14 /10/2024 What is Culture Media? Culture media is a gel or liquid that contains nutrients and is used to grow bacteria or microorganisms. They are also termed growth media. Significance – Culture media is used to identify the causative agent from infected material. characteristic of good culture media: it must contain in proper amount all the ingredients that are need for bacterial growth as: 1-source of nitrogen 8-Indicators 2-source of carbohydrate a-PH indicator which detect the changes in H ion 3-source of mineral concentration due to bacterial metabolism 4-water b- detection of certain product 5-solidifing agent as using of ferric or ferrous ions for the detection of as agar which is polysaccharide extracted from red seaweed algae H2S production melting at 98°C (100 °C) solidifying at 42°C (40 °C) in solid media 20 g/l (1-2%) in semi solid media 2-3 g/l (0.2-0.4%) 6-Enrichement 7-Inhibitors used for selective media which inhibit growth of certain M.O and allow or permit growth of other EX: anilin dyes as brilliant green heavy metal as bithmus chemicals as azide, citrate, and selenite antimicrobial agent as neomycin and vancomycin  Application of culture media For culture and isolation of the causative agent Proper identification through colonies morphological character and biochemical testing. For culture purification and stock preparation. To check antimicrobial agents and preservatives effect. To differentiate between different colonies. To create antigens for laboratory use. To estimate viable count. To test antibiotic sensitivity. Limitations of culture media Risk of cross-contamination. Need complete sterilized area (expensive tool dependent) High skill required for optimal results. Increased drying out of media can occur.  Classification of media Based on the consistency: according to amount of agarose gel used Liquid media Solid media Semi Solid media (broth) natural synthetic mixed Based on the origin from plant or animal from known chemical As mycoplasma origin (potato, compound as selenite media (40% serum + peptone, serum and and tetrathionate chemical) blood) broth Based on the constituent and use 1-simple (ordinary, Basal, 4- 2-Enriched 3-Specific General) Selective 5-Differential 7-Transport media 8-Storage media 6-Indicator 1-Ordinary or simple media - Most commonly used in routine labs. - Known as Basal medium or general medium Uses: For Isolation of most and common bacterial isolates without any restrictions For detection some biochemical characteristics of the isolated pathogens such as (sugar fermentation test and indole test) e.g. Nutrient broth, NA, peptone water, liver infusion broth Disadvantage of liquid media ❖ Not exhibit ch.ch of bacterial growth appearance ❖ Cross contamination: M.O can not be separated from mixture 3-Nutrient agar this media can overcome disadvantage of liquid media this media is better for bacterial isolation &also exhibit ch.ch of colonies easily to be identified Use: for isolation of aerobic & facultative anaerobic 4-Liver infusion broth: For cultivation of brucella  2- Enriched media —-Blood, serum or others added to the basal medium (nutrient broth or agar). —-To support growth of bacteria which are more needing nutritional requirements (fastidious bacteria as strept or corynebacterium). 1-Blood, serum, and glucose -blood and serum (10%) is agar but glucose (10%) is broth -blood must be sterile, from healthy animal or person, collected under aseptic condition and non coagulated by addition of anticoagulant (EDTA, citrate and heparin) 2-Choclate agar (heated blood agar) - heating blood agar in water bath at 75c for 10 min until blood become chocolate brown in color -used for 1- Haemophillus influnza 2- Pnemococci 3-Campylobacter 3- Indicator media These media contain an indicator which changes its color when a bacteria grows in them due to fermentation processes by microorganism to detect any change occur in PH. 1-Sugar media-used to determine the fermentation process of some M.O composition :peptone water +1%sugar+1%a red's indicator ( neutral colorless acidic red) 2-litmus milk used as PH indicator in milk sugar fermentation  4- Selective media Certain chemical substances added to ordinary media or enriched media to inhibit the growth of undesirable M.O and allow the growth of others. Type of media Targeted pathogens Mannitol Salt Agar It has 7% of sodium chloride that inhibits the growth of most microbes and promotes Selective for Staphylococcus aureus the growth of Staphylococci. It has phenol red dye that changes in acid PH from red to yellow (Differential) Salmonella-Shigella Agar Selective for Salmonella (Central black colonies). Deoxycholate agar Selective for Shigella (colorless colonies). MacConkey Agar Selective isolation for Enterobacteriaceae - It has bile salts that inhibit the growth of Gram-positive bacteria TCBS Agar – Thiosulfate-citrate-bile salts-sucrose (TCBS) - Bile salt inhibits the growth of unwanted Gram-positive bacteria Selective and differential for Vibrio cholera, V. - sodium thiosulfate and sodium citrate to inhibit the growth of Enterobacteriaceae parahemolytica and other Vibrio species - Sucrose (sucrose) is included as a fermentable carbohydrate - Thymol blue and bromothymol blue are included as indicators of pH changes used for isolation and enumeration of members of Violet Red Bile Glucose (VRBG) agar the family Enterobacteriaceae in food products.  Type of media Targeted pathogens Enriched, selective, and differential media Baird-Parker agar base - used for the detection of coagulase-positive staphylococci (S. aureus) from food and other non-clinical sources )Black colonies surrounded with hallow zone) Cetrimide agar Selective and differential medium - used for the identification of Pseudomonas aeruginosa Selective differential medium - Inhibits the growth of Gram-positive bacteria and provides a color indicator distinguishing Eosin Methylene Blue (EMB) agar between organisms that ferment lactose (e.g., E. coli) and those that do not (e.g., Salmonella, Shigella). Charcoal selective medium Enriched selective medium - used for the isolation of Campylobacter species b- liquid selective media 2- Tetrathionate broth 1-Selenite f broth(Na selenite) allow growth of salmonella( typhi, parathyphi and inhibit salmonella group coliform group) 1,2 enrichment of culture of salmonella from fecal sample 5-Differential media A media to distinguish between bacteria depending on the diverse metabolic activity of M.O. 1- Blood agar 2- MacConkey agar act as enriched and differential media differentiate between hemolytic and non hemolytic - act as a Selective and differential medium. bacteria based on the production of hemolysin Selective –bile salts inhibitors for most gram positive. enzymes Differentiate- Between Gram-negative bacteria by their ability to ferment lactose. A. Beta hemolysis: Enzymes lyse the blood cells completely, producing a clear area around the colony. Pink colonies: Bacteria produce acid chane the B. Alpha hemolysis: Incomplete hemolysis produces a color due to lactose fermenter ex: E.coli greenish discoloration around the colony. Pale or white colonies- Non fermenters ex: C. Gamma hemolysis: No effect on the red cells. salmonella  6- Specific media 1-media used for cultivation of mycobacteria TB a-lowenstien Jensen media -used to differentiate between bovine and human type of TB by the morphology of colonies on the media Lowenstein Jensen Media- It is made selective by adding malachite green and stops the unwanted growth of pathogens. b-dorest's egg media c-petragenani media d- potato media -used for cultivation of human TB type 2- media for anaerobic cultivation of clostridia 3- media for cultivation of fungi a- cooked meat medium (Robertson's media ) -increasing of sugar and decreasing PH (acidic) ex: sabroud dextrose agar -used in cultivation &preservation of anaerobes all media sterilized by autoclaving except media contain serum,egg and media contain gelatin,milk and glucose  7- Transport media Culture media that are used to maintain the viability of pathogenic microorganisms present in a clinical sample and prevent the potential pathogens to be overgrown by other commensals or during the transportation to the lab. Transport Media Type Intended Use 1-Stuart Medium Semisolid Transportation of swabs for recovery of fastidious, non- fastidious, and anaerobic bacteria 2-Cary-Blair Medium Semisolid Transport of fecal and rectal samples for recovery of enteric pathogens 3-Mycoplasma Transport Broth Liquid medium Transportation of swabs or body fluid for recovery of Mycoplasma, Ureaplasma.  8- Storage media - It’s used to store microorganisms for a longer period Ex: chalk cooked meat broth egg saline medium Serial dilution Spread plate technique: Pour plate technique:  Laboratory purification of bacteria by Streak Plate Methods: A)Continuous Streak: B) Quadrant Streak : Used to obtain a pure culture from the mixed culture in order to perform morphological, biochemical, and molecular tests to identify and for other applications. Used to define the specimen as pure or mixed species.  Bacterial stain &staining one of the most important characteristics of bacteria is their morphology (size, shape, arrangement &structure). this ch.ch can only be determined by examination of properly prepared &stained specimens. Dyes are commercially prepared as salts: (MB)CL+ bacterial cell surface(Na) Bacterial cell surface MB+ NaCl (exchange of -ve and +ve charges) 1-Smear from solid media 2-Smear from liquid media sterilize loop in benzene flame as same steps of solid media without applying a drop of On the center of the dry clean slide place one drop water of distilled water by the loop then resterilize the loop &transfer a loop fill of culture to the Fixation is done by heat in all samples except blood is center of the slide &mix them done by methyl alcohol Mixture is spread (distribute and emulsify) in the middle 2/3 of the slide (not to be too thick or too thin) restrilize the loop to avoid any contamination allow the smear to air dry until looks like a white film heat fixation of smear (passing 2-3 times through flame) to make adherence and fixation of M.O on the slide and kill them. Avoid overheating ,otherwise cheering of film Put a drop of cedar wood oil on the center of the slide &and examine under O.I.L (after staining)  Classification of stain Acc. Acc. To no. of ACC. To it’s Differential stain to PH ingredients Specificity Acidic stain……… 1-Simple stain 1-Capsule demonstration 1-Gram (acid stain(G+VE and G fuchsine) -VE) 2-Basic 2-Compound stain 2-Flagella demonstration 2-Zeil Nelson stain stain………… (Acid &alcohol violet fast and non acid ,methylene &non alcohol blue(MB) fast) 3-Spore demonstration 4-Volatine (metachromatic granules) demonstration  Simple stains Loeffler’s MB (Loffler’s Polychrome MB Leishman’s stain methylene blue) Use: Use: Use: 1-Examination of blood parasite (malaria Staining of bacillus 1-Examination of milk and Trypanosoma) sample for Strept agalactia anthracis(MACFEDIAN 2-Examination of bacterial septicemic REACTION) in film prepared 2- used as a counter stain from blood or tissue of dead disease (Pasteurella which ch.ch by in Zielnelson stain animal bipolarity) capsule stained with purple color and body stained with blue color  Zheil nelson's Differential stains Gram 's stain stain Gram's stain Use: useful for identifying and classifying of bacteria into G+VE and G-VE microorganism Composition 1- crystal violet (1st stain) 2-gram's iodine (mordant) 3- ethyl alcohol 4- safranine(2nd stain) Principle: the target of the stain is bacterial cell wall and its composition differs from G+VE &G-VE bacteria as mgribonuclate(peptidoglycan) present only in cell wall of G+VE bacteria , mg ribonuclate during staining combine with crystal violet gram's iodine complex resulting in formation of insoluble compound in alcohol so G+VE bacteria retain primary stain so appear violet  Ziehl nelson stain use: classification of bacteria into acid &alcohol fast and nonacid &non alcohol fast Composition: 1-strong carbol fuchsin 2-acid alchol soln. 3- loffler M.B or malachite green as counter stain Principle: staining of M.O which is difficult to be stained by ordinary stain (TB) which is bacilli surrounded by wax &lipid envelope so need strong stain and heat to allow the stain penetration, so TB appear as red due to carbol stain occupy receptor of TB waxy envelop glycolipid and peptidoglycolipid TB (acid fast) red other (nonacid fast) blue or green  Specific stain 1-capsular stain wet Indian ink film dry Indian ink film 2-spore staining malachite green staining (rackette's stain)  L.P (low power)= 10 X10 = 100 X Calculate the power of microscope H.P(high power) = 10 X 40 = 400 X Total magnification power of microscope = OIL(oil immersion lens) = 10 X 100 = 1000 X magnification power of eye piece X magnification power of objective lens Very important in bacteriology Examination of bacterial film 1-Rotate the revolving nose piece to get the oil immersion lens in an accurate position for the examination of the slide 2- put a drop of cedar wood oil On the slide 3-put the slide on the stage 4-move the oil immersion lens slowly by course adjustment knob until the oil contacts it 5-use the fine adjustment knob to get the sharp focus NB……During examination by OIL, we add a drop of cedar wood oil due to it has the same refractive index of light in glass, So no reflection Of light occurs. After the end of examination by OIL we clean the lens by piece of gauze soaked in xylol (fat solvent). Avoid using of acid or alcohol because it cause tearing of the lens  ((Micrometry & microbial size)) The unit of measurement of bacterial diameter is Methods of detection of bacterial size:- micron (µ). (types of micrometry) *classification of bacteria according to size:- 1- ocular and stage method small size (1-2 micron) Pasteurella 0.2-micron 2- photographic method Brucella 1 micron 3- Electron microscopical method medium size (2-4 micron) E.coli, Salmonella, Corynebacterium large size (4-10 micron) Bacillus anthracis , TB(Mycobacterium tuberculosis). very large (filamentous) Spirochaetes (100 micron) * bacterial motility* 2 types of motility: Types of bacteria acc. To motility false or Brownian motility(tumbling movement): Non motile bacteria Def: it is movement due to vibration of particles of Ex : Staph , Strept, Corynebacterium, Shigella , the media. Or due to continuous bombardment of Klebsiella molecules of the media. Motile bacteria true motility: Have the ability to move forward and backward Ex: The ability of bacterial cell to go forward and E.coli , proteus , Bacillus anthracoid , all backward by organs of locomotion. Salmonella except S.gallinarum (pullorum disease) All Clostridium except C.perfrengens Classification of motile bacteria (types of motility of motile bacteria) a- flagellar motility b- spirochaetal motility a- flagellar motility atrichus → no flagella. monotrichus → one flagella at one side. amphitrichus → two flagella one at each side. lophotrichus → band of flagella at one side or both sides. peritrichus (holotrichus) → flagella all over the bacteria b- spirochetal motility (cell body movement) due to the presence of axial filament in periplasmic space all over the body. the movement by contraction , relaxation of axial filament (contractile filament). *Methods of detection of bacterial motility: 1-hanging drop technique 2-cultivation of semisolid agar or U-tube method 3-swarming on solid media 1- Hanging drop technique *Requirements -glass slide with concavity-cover slide-young broth culture *Procedure: put a small amount of Vaseline around the concavity of the slide. put drop of culture on the cover slide invert the slide with concavity on the cover slide and quickly turn around then examine under high power X40. * significance of Vaseline: Prevent air dryness-Prevent air movement-Prevent liquid spreading 2. Cultivation on semisolid agar or U-tube method In U shaped tube or in normal tube make inoculation by pointed loop till the middle of the tube (in U shaped tube stabbing at one side). Incubation at 37 °C , examine at 6, 12,18,24 hours bacteria spread away from site of inoculation (motile) growth of bacteria only at site of inoculation (non motile). the turbidity transfer to the other side of the tube. N.B *The growth of bacteria in liquid media appear as turbidity while growth of bacteria on solid media appear as colonies. *why we use semisolid media for detection of motility? Because it allow diffuse spreading growth and turbidity in case of motile bacteria that can recognized by naked eye. 3. Swarming on solid media It is character of highly active motile bacteria (Proteus) which appear as thin successive waves of growth on the surface of the media. Swarming definition is the ability highly active motile bacteria such as Proteus to spread in a thin film and successive waves which cover the whole surface of solid media.  Assignment Culture media type use stain type use Explain the importance of cedar wood oil during examination of bacterial film. Classification of bacterial motility. Discuss methods of detection of bacterial motility.